Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia (PCP) in humans which remains a leading opportunistic infection associated with AIDS patients, even in the era of Highly Active Anti-Retroviral Therapy (HAART). PCP increasingly targets new groups of patients with underlying chronic disease states, such as patients receiving anti-TNF therapy, other immunosuppressive agents and with underlying chronic diseases such as Chronic Obstructive Pulmonary Disorder (COPD). The combination therapy of trimethoprim- sulfamethoxazole (TMP-SMX) remains the standard prophylactic and therapeutic modality in use today with few alternative treatments. Evidence for mutations associated with resistance to sulfamethoxazole has been identified in the P. jirovecii dihydropteroate synthase encoding gene. It is thus critical that new approaches to anti-PCP therapy be developed. In this proposal, we will build on our previous observations that slightly acidified nitrite (A-NO2-), a nitric oxide (NO) generator, was effective in preventing the formatio of in vitro biofilms by Pneumocystis carinii and decreased viability in standard suspension cultures. Ongoing toxicology studies in dogs and rats and Phase I human trials revealed A-NO2- was well tolerated at doses up to 19-20 mg/kg, respectively, with minimal side effects, which bodes well for translation of these efforts. We propose to evaluate 2 different delivery modes for A-NO2- in therapeutic and prophylaxis mouse models of PCP at 3 different doses to determine the most effective administration route and treatment times that reduce fungal lung burdens. Initial immunological studies will guide further mechanistic experiments to determine the mode of action of this new alternative agent.